Theorell et al: Unperturbed cytotoxic lymphocyte phenotype and function in ME/CFS

mango

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Unperturbed cytotoxic lymphocyte phenotype and function in myalgic encephalomyelitis/chronic fatigue syndrome patients

Jakob Theorell1*, Indre Bileviciute Ljungar2, 3, Bianca Tesi4, 5, Heinrich Schlums1, Mette S. Johnsgaard6, Babak Asadi Azarbaijani7, 8, Elin Bolle Strand7, 9 and Yenan T. Bryceson1, 10*
  1. Department of Medicine, Huddinge, Karolinska Institutet, Sweden
  2. Department of Rehabilitation Medicine, Karolinska Institutet, Sweden
  3. Department of Clinical Sciences, Danderyd, Karolinska Institutet, Sweden
  4. Department of Women’s and Children’s Health, Karolinska Institutet, Sweden
  5. Department of Molecular Medicine and Surgery, Karolinska Institutet, Sweden
  6. Klinikk for Alle, Storo, Norway
  7. Department of Geriatric Medicine, Oslo University, Norway
  8. VID Specialized University, Norway
  9. Department of Geriatric Medicine, Oslo University, Norway
  10. Department of Clinical Science, University of Bergen, Norway
Front. Immunol. | doi: 10.3389/fimmu.2017.00723
Received: 15 Dec 2016; Accepted: 08 Jun 2017.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a debilitating disorder linked to diverse intracellular infections as well as physiological stress.

Cytotoxic lymphocytes combat intracellular infections. Their function is attenuated by stress.

Despite numerous studies, the role of cytotoxic lymphocytes in ME/CFS remains unclear.

Prompted by advances in the understanding of defects in lymphocyte cytotoxicity, the discovery of adaptive NK cell subsets associated with certain viral infections, and compelling links between stress, adrenaline and cytotoxic lymphocyte function, we re-assessed the role of cytotoxic lymphocytes in ME/CFS.

Forty-eight patients from two independent cohorts fulfilling the Canada 2003 criteria for ME/CFS were evaluated with respect to cytotoxic lymphocyte phenotype and function. Results were compared to values from matched healthy controls.

Reproducible differences between patients and controls were not found in cytotoxic lymphocyte numbers, cytotoxic granule content, activation status, exocytotic capacity, target cell killing or cytokine production.

One patient expressed low levels of perforin, explained by homozygosity for the PRF1 p.A91V variant. However, overall, this variant was present in a heterozygous state at the expected population frequency among ME/CFS patients.

No single patient displayed any pathological patterns of cellular responses. Increased expansions of adaptive NK cells or deviant cytotoxic lymphocyte adrenaline-mediated inhibition were not observed.

In addition, supervised dimensionality reduction analyses of the full, multidimensional datasets did not reveal any reproducible patient/control discriminators.

In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide useful biomarkers for the diagnosis of ME/CFS.

http://journal.frontiersin.org/article/10.3389/fimmu.2017.00723/abstract
 

mango

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Please note that isolated NK cells were used in this study, unlike in many other previous studies. Which means that if it's true that there's something in the blood of pwme that negatively affects the cells (as suggested by for example Drs Ron Davis and Fluge & Mella), the method the researchers chose for this particular study might have affected the result.

I believe this issue was discussed during the Invest in ME conference in London recently? Please correct me if I'm wrong, or add more info if you can, please :)
 

mango

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OverTheHills blog post said:
Dr. Jakob Theorell is on the MD-PhD program at the Karolinska Institute. He presented on his two-centre Norwegian/Swedish study looking at NK cells and cytotoxic T cells in ME patients.

He started with a quick immune 101 review — (that works for me!) — T cells are part of the adaptive immune system, they have the individual’s memory of pathogens, developed over a lifetime. NK cells are part of the innate immune system, pre-programmed with “species memory” of immune threats. So for these types of cell, what they do is similar (killing foreign cells) and they use a similar mechanism. They also release pro-inflammatory cytokines.

For these cells to do their job properly, they need to have their perforins and granzymes present and able to be released. If they are able to latch onto a pathogen but not kill it because of some impairment, you get what Jakob described as “immune frustration,” where there is more and more signalling without the ability to kill the intruder. The whole immune system spins out of control.

For a long time, medicine has recognised genetic diseases of cytotoxic lymphocytes (sometimes caused by a single mutation) which are expressed soon after birth and are fatal if not treated. But now researchers are identifying and studying more subtle immune impairments which can reveal themselves later in life. Jakob's hypothesis is that a substantial subset of people with ME have impaired lymphocyte toxicity, that they are unable to deal with pathogens (or perhaps other immune insults??) properly.

Jakob went on to describe some, perhaps controversial, negative results. He found no significant difference between patients and controls in their levels of perforins and granzymes, nor with cytotoxic granule release under his test conditions. Surprisingly, there was no difference in cell-killing ability either. But like a good researcher does, he started looking at why this might be (rather than inventing myths about false illness beliefs).

Perhaps the freezing, thawing and resting of cells before testing had differentially killed off “sick” or impaired NK cells, leaving only normal ones which would behave ... well ... normally. But there was no difference in the percentage of NK cells between ME patients and controls, so this seems unlikely.

Perhaps some serum factor makes the NK cells functionally incompetent in patients, but not when cells have been separated out. Jakob didn’t test for this, but recognises the importance of this for future experiments (and I think it was Mary Ann Fletcher who reinforced the importance of whole blood assays in the Q&A session). There are many candidates for the serum factor in Jakob's view, as NK cells have a wide variety of receptors (including lactate and adrenaline) and are regulated by many things.

He speculated that adrenaline could be an important factor here. Adrenaline is known to be important in inhibiting NK cells, and getting to a doctor’s surgery/office is such a major effort for ME patients they are likely to be running on adrenaline or, as he put it, having a major catecholamine event.

It’s well known that ME patients have HPA dysfunction and he did find one difference in his experiments — the ME patients' cells were less responsive to adrenaline. This is only a small study, but he suggested this is a worthwhile angle to follow up.

Personal Commentary: Jakob spoke fast and had quite a strong Norwegian accent (there was some banter about him being Norwegian and his supervisor Swedish, so I hope that's right). But I got the impression that he was both a clear thinker and a good communicator. I enjoyed his presentation a lot, despite it being right at the limit of what I (as an absolute lay person) can understand.

It’s really important that young researchers get ahead in this field, and as other presenters also pointed out during the conference, negative results are important and can tell us much, not least about methodology. So well done, Jakob!
http://phoenixrising.me/archives/28424

http://forums.phoenixrising.me/inde...st-in-me-conference-part-1.52321/#post-865502
 

msf

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Would the lack of difference with controls suggest that these patients did not have intracellular infections? I can´t seem to access the full article to see the authors´ conclusions.
 

Manganus

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Kudos, @mango !

Often, we focus too much on positive findings and forget articles that don't show a result.
That's understandable, psychologically, but nevertheless unfortunate.

In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide useful biomarkers for the diagnosis of ME/CFS.
 

Anne

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From the article:

"Our negative results contrast a number of previously published studies indicating diminished NK cell function in ME/CFS (for a comprehensive list of relevant publications overall content and outcome, see Table S4 in Supplementary Material). A few studies have shown that K562 cell killing was diminished in whole-blood assays (13, 37). Other studies found similarly diminished NK cell cytotoxic activity using PBMC immediately after isolation (15, 46), whereas other investigators have not found any impairments of NK cell cytotoxic activity using isolated PBMC (16, 62). Timing of functional assays vis-à-vis PBMC isolation may explain differences, with an effect of soluble factors, such as cytokines, catecholamines, and hormones, waning with time from cell isolation from whole blood. Such a discrepancy would therefore suggest that NK cells from ME/CFS patients in general are intrinsically normal but might be responding to an abnormal external milieu, rendering them hypofunctional in whole blood or immediately after isolation. Congruently, the addition of adrenaline, as shown above, did not attenuate NK cell functionality more in the patients than in the controls. If anything, the opposite was observed, which could hypothetically be explained by the presence of β2-adrenergic receptor autoantibodies (63), or downregulations of β2-adrenergic receptors due to an increased tonic adrenergic signaling in the ME/CFS patients. Regardless, our results indicate that cell intrinsic differences in cytotoxic lymphocyte differentiation or function do not clearly distinguish ME/CFS patients from controls."

So they are concluding that when isolated NK cells are studied their function is no different in ME patients, but as overthehill said in his/her blog post: "Perhaps some serum factor makes the NK cells functionally incompetent in patients, but not when cells have been separated out."

I am disappointed that they didn't extend the study to do a whole blood analysis in parallel with analysis of isolated NK cells. As I understand it, they would have made a breakthrough in the ME field if they had been able to prove that NK cell function is lowered in ME in whole blood or serum, but not in isolated NK cells. I am very much hoping now that someone else will do that experiment. Ron Davis' team? @Janet Dafoe (Rose49) @Ben Howell One of the teams cooperating with Invest in ME Research in the UK? The CDC, who will be studying NK cells as a part of their multisite study?

@Janet Dafoe (Rose49) @Jonathan Edwards , I would love to hear your comments!

@alex3619 @alicec @mango FYI
 
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I don't believe the low NK Cell activity found in other studies is a causative factor in ME or CFS, it is just a secondary consequence.
The explanation provided in Anne's post above seems plausible, though I hypothesise it is dysfunction of other GPCR, rather than β2-adrenergic receptors specifically.
 

Anne

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From the article:
I am disappointed that they didn't extend the study to do a whole blood analysis in parallel with analysis of isolated NK cells. As I understand it, they would have made a breakthrough in the ME field if they had been able to prove that NK cell function is lowered in ME in whole blood or serum, but not in isolated NK cells. I am very much hoping now that someone else will do that experiment. Ron Davis' team? @Janet Dafoe (Rose49) @Ben Howell One of the teams cooperating with Invest in ME Research in the UK? The CDC, who will be studying NK cells as a part of their multisite study?
To take this issue forward I have emailed Elizabeth Unger at the CDC (no reply yet) and Avindra Nath at the NIH. For Dr Nath's response, see below.

I would also love to hear if a comparison of NK cell function in blood vs in isolated cells is something the Stanford/OMF team could include in the Severely Ill Study? @Ben Howell @Janet Dafoe (Rose49)

Från: Nath, Avindra (NIH/NINDS)
Skickat: den 27 juli 2017 21:47
Till: Anne Örtegren
Ämne: Re: Question regarding the NIH Intramural ME/CFS study

Dear Anne,
Thank you for bringing this to my attention. We will be glad to look into this further.
All the best.
Avi

From: Anne Örtegren
Date: Wednesday, July 26, 2017 at 11:44 AM
To: "Nath, Avindra (NIH/NINDS) [E]"

Dear Dr Nath,

Firstly, let me thank you for your work with the ME/CFS intramural study! It brings hope.

I’m sure you have seen the following paper on NK cell function in ME/CFS from Karolinska in Stockholm, which was also discussed at the Invest in ME Research conference in London: http://journal.frontiersin.org/.../fimmu.2017.00723/full

They conclude that when isolated NK cells are studied their function is no different in ME patients, but they note that other studies using whole blood have shown lower NK cell function. This led them to discuss in London the possibility that there might be some serum factor making the NK cells functionally incompetent in patients, while isolated cells which have been separated out function normally. This seems to chime in with findings from the Fluge and Mella group and the Ron Davis group indicating that some serum factor is inhibiting cell function in ME.

As I understand it, a study would now need to be done which uses blood from a group of well-defined ME patients and performs parallel analyses: NK cell function in whole blood or serum and NK cell function in isolated NK cells.

If it could thus be shown that NK cell function is normal in isolated cells but abnormal in whole blood or serum, we would have taken a large step towards the understanding of the disease mechanisms in ME, wouldn’t we? It would tell us that a serum factor is in fact at play, and that NK cell function is indeed low in ME patients’ blood.

It seems to me extremely important that this is followed up. Unfortunately as I understand it the Stockholm group aren’t planning any further studies, so it is up to other research groups to pursue this important lead.

Would you include this seemingly potentially ground-breaking comparison between whole blood analysis of NK cell function and analysis of function of isolated NK cells as a part of your intramural study at the NIH?

Thank you in advance.

Best wishes,
Anne Ortegren

I would also love to hear if @Jonathan Edwards or @charles shepherd have any comments.
 

Ben H

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To take this issue forward I have emailed Elizabeth Unger at the CDC (no reply yet) and Avindra Nath at the NIH. For Dr Nath's response, see below.

I would also love to hear if a comparison of NK cell function in blood vs in isolated cells is something the Stanford/OMF team could include in the Severely Ill Study? @Ben Howell @Janet Dafoe (Rose49)

Från: Nath, Avindra (NIH/NINDS)
Skickat: den 27 juli 2017 21:47
Till: Anne Örtegren
Ämne: Re: Question regarding the NIH Intramural ME/CFS study

Dear Anne,
Thank you for bringing this to my attention. We will be glad to look into this further.
All the best.
Avi

From: Anne Örtegren
Date: Wednesday, July 26, 2017 at 11:44 AM
To: "Nath, Avindra (NIH/NINDS) [E]"

Dear Dr Nath,

Firstly, let me thank you for your work with the ME/CFS intramural study! It brings hope.

I’m sure you have seen the following paper on NK cell function in ME/CFS from Karolinska in Stockholm, which was also discussed at the Invest in ME Research conference in London: http://journal.frontiersin.org/.../fimmu.2017.00723/full

They conclude that when isolated NK cells are studied their function is no different in ME patients, but they note that other studies using whole blood have shown lower NK cell function. This led them to discuss in London the possibility that there might be some serum factor making the NK cells functionally incompetent in patients, while isolated cells which have been separated out function normally. This seems to chime in with findings from the Fluge and Mella group and the Ron Davis group indicating that some serum factor is inhibiting cell function in ME.

As I understand it, a study would now need to be done which uses blood from a group of well-defined ME patients and performs parallel analyses: NK cell function in whole blood or serum and NK cell function in isolated NK cells.

If it could thus be shown that NK cell function is normal in isolated cells but abnormal in whole blood or serum, we would have taken a large step towards the understanding of the disease mechanisms in ME, wouldn’t we? It would tell us that a serum factor is in fact at play, and that NK cell function is indeed low in ME patients’ blood.

It seems to me extremely important that this is followed up. Unfortunately as I understand it the Stockholm group aren’t planning any further studies, so it is up to other research groups to pursue this important lead.

Would you include this seemingly potentially ground-breaking comparison between whole blood analysis of NK cell function and analysis of function of isolated NK cells as a part of your intramural study at the NIH?

Thank you in advance.

Best wishes,
Anne Ortegren

I would also love to hear if @Jonathan Edwards or @charles shepherd have any comments.
Hi @Anne

Will try to find out for you. It may well have already been done.


B